A Multiplexed, Confinable CRISPR/Cas9 Gene Drive Can Propagate in Caged Aedes Aegypti Populations

Overview

Journal Nat Commun

Specialty Biology

Date 2024 Jan 25

PMID 38272895

Authors

Michelle A E Anderson

Estela Gonzalez

Matthew P Edgington

Joshua X D Ang

Deepak-Kumar Purusothaman

Lewis Shackleford

Katherine Nevard

Sebald A N Verkuijl

Timothy Harvey-Samuel

Philip T Leftwich

Kevin Esvelt

Luke Alphey

Affiliations

Soon will be listed here.

Abstract

Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.

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References

Li M, Yang T, Kandul N, Bui M, Gamez S, Raban R . Development of a confinable gene drive system in the human disease vector . Elife. 2020; 9. PMC: 6974361. DOI: 10.7554/eLife.51701. View

Anderson M, Purcell J, Verkuijl S, Norman V, Leftwich P, Harvey-Samuel T . Expanding the CRISPR Toolbox in Culicine Mosquitoes: Validation of Pol III Promoters. ACS Synth Biol. 2020; 9(3):678-681. PMC: 7093051. DOI: 10.1021/acssynbio.9b00436. View

Kandul N, Liu J, Buchman A, Gantz V, Bier E, Akbari O . Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes. G3 (Bethesda). 2019; 10(2):827-837. PMC: 7003086. DOI: 10.1534/g3.119.400985. View

Champer J, Yang E, Lee E, Liu J, Clark A, Messer P . A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population. Proc Natl Acad Sci U S A. 2020; 117(39):24377-24383. PMC: 7533649. DOI: 10.1073/pnas.2004373117. View

Ang J, Nevard K, Ireland R, Purusothaman D, Verkuijl S, Shackleford L . Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source. PLoS Genet. 2022; 18(2):e1010060. PMC: 8893643. DOI: 10.1371/journal.pgen.1010060. View

Bottino-Rojas V, Ferreira-Almeida I, Nunes R, Feng X, Pham T, Kelsey A . Beyond the eye: Kynurenine pathway impairment causes midgut homeostasis dysfunction and survival and reproductive costs in blood-feeding mosquitoes. Insect Biochem Mol Biol. 2022; 142:103720. PMC: 11055609. DOI: 10.1016/j.ibmb.2022.103720. View

Akbari O, Antoshechkin I, Amrhein H, Williams B, DiLoreto R, Sandler J . The developmental transcriptome of the mosquito Aedes aegypti, an invasive species and major arbovirus vector. G3 (Bethesda). 2013; 3(9):1493-509. PMC: 3755910. DOI: 10.1534/g3.113.006742. View

Anderson M, Mavica J, Shackleford L, Flis I, Fochler S, Basu S . CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say. PLoS One. 2019; 14(11):e0224857. PMC: 6850532. DOI: 10.1371/journal.pone.0224857. View

Noble C, Olejarz J, Esvelt K, Church G, Nowak M . Evolutionary dynamics of CRISPR gene drives. Sci Adv. 2017; 3(4):e1601964. PMC: 5381957. DOI: 10.1126/sciadv.1601964. View

10.

Han Q, Calvo E, Marinotti O, Fang J, Rizzi M, James A . Analysis of the wild-type and mutant genes encoding the enzyme kynurenine monooxygenase of the yellow fever mosquito, Aedes aegypti. Insect Mol Biol. 2003; 12(5):483-90. PMC: 2629591. DOI: 10.1046/j.1365-2583.2003.00433.x. View

11.

Gerdes J, Mannix K, Hudson A, Cooley L . HtsRC-Mediated Accumulation of F-Actin Regulates Ring Canal Size During Oogenesis. Genetics. 2020; 216(3):717-734. PMC: 7648574. DOI: 10.1534/genetics.120.303629. View

12.

Guichard A, Haque T, Bobik M, Xu X, Klanseck C, Kushwah R . Efficient allelic-drive in Drosophila. Nat Commun. 2019; 10(1):1640. PMC: 6456580. DOI: 10.1038/s41467-019-09694-w. View

13.

Pham T, Phong C, Bennett J, Hwang K, Jasinskiene N, Parker K . Experimental population modification of the malaria vector mosquito, Anopheles stephensi. PLoS Genet. 2019; 15(12):e1008440. PMC: 6922335. DOI: 10.1371/journal.pgen.1008440. View

14.

Purusothaman D, Shackleford L, Anderson M, Harvey-Samuel T, Alphey L . CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say. Sci Rep. 2021; 11(1):14964. PMC: 8298393. DOI: 10.1038/s41598-021-94065-z. View

15.

Maselko M, Feltman N, Upadhyay A, Hayward A, Das S, Myslicki N . Engineering multiple species-like genetic incompatibilities in insects. Nat Commun. 2020; 11(1):4468. PMC: 7478965. DOI: 10.1038/s41467-020-18348-1. View

16.

Alphey L, Crisanti A, Randazzo F, Akbari O . Opinion: Standardizing the definition of gene drive. Proc Natl Acad Sci U S A. 2020; 117(49):30864-30867. PMC: 7733814. DOI: 10.1073/pnas.2020417117. View

17.

Martins S, Naish N, Walker A, Morrison N, Scaife S, Fu G . Germline transformation of the diamondback moth, Plutella xylostella L., using the piggyBac transposable element. Insect Mol Biol. 2012; 21(4):414-21. DOI: 10.1111/j.1365-2583.2012.01146.x. View

18.

Anderson M, Gonzalez E, Ang J, Shackleford L, Nevard K, Verkuijl S . Closing the gap to effective gene drive in Aedes aegypti by exploiting germline regulatory elements. Nat Commun. 2023; 14(1):338. PMC: 9860013. DOI: 10.1038/s41467-023-36029-7. View

19.

Akbari O, Matzen K, Marshall J, Huang H, Ward C, Hay B . A synthetic gene drive system for local, reversible modification and suppression of insect populations. Curr Biol. 2013; 23(8):671-7. PMC: 8459379. DOI: 10.1016/j.cub.2013.02.059. View

20.

Noble C, Min J, Olejarz J, Buchthal J, Chavez A, Smidler A . Daisy-chain gene drives for the alteration of local populations. Proc Natl Acad Sci U S A. 2019; 116(17):8275-8282. PMC: 6486765. DOI: 10.1073/pnas.1716358116. View